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Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment

Author

Listed:
  • Scott A. Robson

    (Harvard Medical School)

  • Koh Takeuchi

    (National Institute of Advanced Industrial Science and Technology)

  • Andras Boeszoermenyi

    (Harvard Medical School
    Dana-Farber Cancer Institute)

  • Paul W. Coote

    (Harvard Medical School)

  • Abhinav Dubey

    (Harvard Medical School
    Dana-Farber Cancer Institute)

  • Sven Hyberts

    (Harvard Medical School)

  • Gerhard Wagner

    (Harvard Medical School)

  • Haribabu Arthanari

    (Harvard Medical School
    Dana-Farber Cancer Institute)

Abstract

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαβ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.

Suggested Citation

  • Scott A. Robson & Koh Takeuchi & Andras Boeszoermenyi & Paul W. Coote & Abhinav Dubey & Sven Hyberts & Gerhard Wagner & Haribabu Arthanari, 2018. "Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-017-02767-8
    DOI: 10.1038/s41467-017-02767-8
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